Yersinia pestis has a set of virulence proteins encoded by plasmid pCD1. Expression and secretion of these proteins are regulated by environmental temperature and Ca2+ through a mechanism called the low-Ca2+ response (LCR). This proposal concerns both the LCR regulatory mechanism and the pathogenic mechanism of one of the virulence proteins called V antigen (LcrV). Aim 1 will determine how LcrV functions in the absence of Ca2+ to prevent downregulation of virulence gene expression. The investigators will use standard molecular genetic approaches to study the gene that encodes a putative small protein that can be crosslinked to LcrV in the absence of Ca2+. They will test a model for how LcrV works by characterizing LCR regulation when known LCR components are varied in expression from regulatable promoters. The regulation in double mutants will determine where in the negative regulatory pathway LcrV has its effect. To identify components with which LcrV interacts, they will obtain and map second-site suppressor mutations of point mutations in LcrV. Aim 2 will develop a model for LcrV's mechanism as a virulence protein and for how anti-LcrV antibody protects mice against experimental plague. They will determine if anti-LcrV interferes with secretion of LcrV, whether LcrV can act alone to modulate a Y. pestis infection, when LcrV works during an infection, and whether it inhibits clearance or phagocytosis of yersiniae in vivo. The investigators will determine whether LcrV decreases inflammation by altering the spectrum of inflammatory cells or cytokines elicited by an infection, and whether LcrV interferes with activation of NK cells or macrophages. Aim 3 will determine how LcrG exerts its regulatory role in the LCR by characterizing LcrG's trafficking during the regulatory response and by identifying LCR components with which LcrG interacts by chemical crosslinking and by mapping point mutations that suppress mutations in IcrG. Aim 4 will develop a model for LcrH's role in LCR repression. They will identify components that LcrH interacts with by chemical crosslinking and by analyzing the regulation in mutant Y. pestis. A genetic approach will be used to identify a hypothetical co-repressor LCR component. These studies will address the 4-decades-old question of how a major Yersinia virulence property functions in pathogenesis. This will be useful in efforts to develop a subunit plague vaccine, as LcrV is a protective antigen and a likely vaccine component. The proposed work also will provide better understand of how the LCR regulation works. This regulation is likely to be novel and to provide a useful paradigm which will facilitate studies of virulence-regulatory mechanisms in other important human pathogens such as Salmonella and Shigella that share some aspects of the LCR mechanism.